Arrays of binding agents or probes, such as polypeptide and nucleic acids, have become an increasingly important tool in the biotechnology industry and related fields. These binding agent arrays, in which a plurality of probes are positioned on a solid support surface in the form of an array or pattern, find use in a variety of different fields, e.g., genomics (in sequencing by hybridization, SNP detection, differential gene expression analysis, identification of novel genes, gene mapping, finger printing, etc.) and proteomics.
In using such arrays, the surface bound probes are contacted with molecules or analytes of interest, i.e., targets, in a sample. Targets in the sample bind to the complementary probes on the substrate to form a binding complex. The pattern of binding of the targets to the probe features or spots on the substrate produces a pattern on the surface of the substrate and provides desired information about the sample. In most instances, the targets are labeled with a detectable label or reporter such as a fluorescent label, chemiluminescent label or radioactive label. The resultant binding interaction or complexes of binding pairs are then detected and read or interrogated, for example by optical means, although other methods may also be used depending on the detectable label employed. For example, laser light may be used to excite fluorescent labels bound to a target, generating a signal only in those spots on the substrate that have a target, and thus a fluorescent label, bound to a probe molecule. This pattern may then be digitally scanned for computer analysis.
Generally, in discovering or designing probes to be used in an array, a nucleic acid sequence is selected based on the particular gene of interest, where the nucleic acid sequence may be as great as about 60 or more nucleotides in length or as small as about 25 nucleotides in length or less. From the nucleic acid sequence, probes are synthesized according to various nucleic acid sequence regions, i.e., subsequences, of the nucleic acid sequence and are associated with a substrate to produce a nucleic acid array. As described above, a detectably labeled sample is contacted with the array, where targets in the sample bind to complimentary probe sequences of the array.
As is apparent, a key step in designing arrays is the selection of a specific probe or mixture of probes that may be used in the array and which maximize the chances of binding with target in a sample. A number of probe design protocols have been developed. For example, probe design may be performed experimentally or computationally.
When designing a genome scanning microarray, it is desirable to provide probes to all transcripts (mRNA sequences) known to occur in the organism in question. There are typically several alternative transcripts produced for each gene; such transcripts typically differ by the inclusion or exclusion of one or more exons, and by the position of the poly-adenylation site (which effectively changes the length of the 3′ most exon). When multiple transcripts are present, it can be difficult to find unique probes for each transcript, particularly with the additional constraints imposed by the need to select for sensitivity and specificity.
There is a need, therefore for the development of probe design protocols that allow one to design a single probe to hybridize to all the alternative transcripts, or at least a portion of the alternative transcripts, for a gene.
Relevant Literature
U.S. patents of interest include: U.S. Pat. Nos. 6,251,588 and 5,556,749. Also of interest is Hosaka et al., Genome Informatics (2001) 12: 449–450.